The importance of post-translational modifications (PTMs) in regulating the global proteome of the eukaryotic cell cannot be underestimated. Ubiquitin (Ub) and Ub-like proteins (Nedd8, SUMO, ISG15, etc.) play a particularly key role for the host. Depending on the topology of the conjugation, these proteins stabilize members of signaling pathways as well as target proteins for destruction by the proteasome. Removal of Ub by host deubiquitinating enzymes (DUBs) are used by eukaryotes to ensure proper regulation of signaling cascades. Modification of these regulatory pathways on the protein-protein level by bacteria represents a key pathogenic target for promoting infection. ChlaDUB1 and ChlaDUB2 are two virulence factors produced by Chlamydia tra- chomatis which act as deubiquitinating and deneddylating enzymes. As Chlamydia does not possess the cellular machinery for ubiquitination of their proteins, it is anticipated that ChlaDUB1 and ChlaDUB2 target the host proteome directly. Indeed, the presence of functional DUBs in the C. trachomatis genome suggest an important role in virulence. Therefore, it is predicted that ChlaDUB1 and ChlaDUB2 directly disrupt the regulation of host immune pathways at the protein-protein level. In order to test this hypothesis, two simultaneous approaches will be used. First, a series of biochemical in vitro assays will be used to dissect the nature of the ChlaDUB1 and ChlaDUB2 substrates and to identify the precise host targets of ChlaDUB1 and ChlaDUB2. Both enzymes are known to sharply attenuate the NF-kB pathway. Experiments are designed to identify the precise Ub-tagged NF-kB pathway members that are targeted by ChlaDUB1 and ChlaDUB2. Second, recognizing that ChlaDUB1 and ChlaDUB2 may target host proteins beyond those represented in the NF-kB pathway, a global screen for targets is required. As a transcriptional analysis is unable to identify changes on the critical level of post-translational modifications, an innovative global protein stability (GPS) assay will be used. An additional advantage of this approach is that can applied back into eukaryotic systems to advance the ubiquitin field as a whole. ! !